1. COBAS AmpliPrep/Cobas TaqMan HIV-1 Test
By: Mary Jo Dowling
July 20,2009
OBJECTIVES:

2. OBJECTIVES:
The test is a Nucleic Acid amplification of HIV type 1, RNA in human plasma.
An automated test developed to test HIV in a single multiplex assay.
It uses real time PCR to detect HIV type 1 group M.
It uses fluorescence to detect HIV-1 target RNA.
The fluorescence is measured by intensity emission forming critical threshold level.

3. RNA Marker Testing and Primers
The detection of HIV-1 target RNA and HIV-1 QS RNA.
The Master Mix reagent has primers and probe specific to HIV-1 and HIV-1 QS RNA.
The Master Mix is developed to have quantitation of HIV subtype group M and fluorescent labeled oligonucleotide probes. It also has a upstream and down stream primers to GAG region of HIV-1.
The HIV-1 QS has HIV-1 RNA containing HIV-1 primer binding sequences and a probe binding region.

4. Clinical Significance of Marker
The HIV-1 QS contains fragments of HIV-1 sequences with primer binding regions identical to HIV-1 target sequence. Then it generates a amplification product,the same as HIV-1 target RNA.
The detection probe binding region of HIV-1 QS states the difference of HIV-1 QS amplicon from HIV-1 target amplicon.
In the annealing process of PCR, the specimens are illuminated and filtered for fluorescence emission.

5. Principle of Procedure
The HIV-1 test is based on specimen preparation to isolate HIV-1 RNA.
It then has Reverse transcription of target RNA to form cDNA.
The next step is simultaneous PCR amplification of target cDNA and detection of HIV-1 target RNA and HIV-1 quantitation standard (QS) RNA, which is probe specific to target.

6. Principle of Procedure -2
Real-time PCR assays can improve the efficiency of end-point PCR tests by providing higher throughput with automation.
End Point is detected by difference in fluorescences of the HIV-1 QS from the HIV-1 target.

7. Extraction Method-1
Specimen Preparation- HIV-1 virus is lysed by incubation at elevated temperature with protease and magnetic glass particles and lysis/binding buffer that release nucleic acid and protects the released HIV-1RNA from Rnases in plasma.
The HIV-1 and HIV-1QS are bound to the glass particles, and waste is washed off.

8. Extraction Method-2
The processed specimen is added to the amplification mixture and transferred to analyzer. The HIV-1 RNA and HIV-1QS RNA are reverse transcribed, amplified and simultaneously detected by cleavage of a target specific and a QS specific dual labeled oligonucleotide probe.
The reverse transcription and amplification reaction is preformed by enzyme Thermus specie, with Manganese and buffer condition allows the reverse transcription and PCR amplification to occur at the same time.

9. Amplification Method-1
The reaction mixture is heated to allow a down stream primer to anneal to HIV-1 RNA and HIV-1 QS RNA. The polymerase extends the annealed primers forming a cDNA to RNA target.
Then the Thermal Cycler in the analyzer heats the reaction mixture to denature RNA/cDNA hybrid and expose the target sequence. The mixture cools, the primers anneal to target DNA.

10. Amplification Method-2
The mixture with all ingredients then extends the annealed primers of target template to produce dsDNA which is the amplicon.
The analyzer then repeats process to increase amplicons only in the region of HIV-1 genome between the primers.

11. Amplification Method-3
The AmpErase enzyme is used for selective amplification of target nucleic acid. The AmpErease is in the master mix and it catalysis the cleavage of DNA opening the chain at the C1 position. Heated in the thermal Cycler, the amplicon DNA chain breaks making the DNA non ampilfiable. The AmpErase remain inactive in 55*C, so it does not destroy target amplicon.

12. Detection Method-1
Detection of PCR products uses RT PCR. The use of dual label fluorescence probes allow for accumulation of product to measure the intensity of the fluorescence emitted by the dyes release during the amplification process.
HIV-1 and HIV-1QS specific oligonucleotide probes has reporter dye and quencher dye which both are labeled with (different fluorescent dye). The dyes are release and separated, and the fluorescence of the reporter dye is increase.

13. Detection Method-2
The amplification of the HIV-1 RNA and HIV-1 QS RNA are measured by intensity of dye. The process is repeated to increase emission intensity.
The fluorescence readings are processed to generate ct values (critical threshold values).

14. Limitation of Procedures-1
Only use human plasma collected in EDTA anticoagulant.
Only test specimens of HIV-1, M group and not used in HIV-1 group O and N nor HIV-2.
Best results depends on specimen processing, collection transport,and storage.
Contamination of HIV-1 positive control must be avoided.
Only personnel train in techniques of PCR

15. Limitation of Procedures-2
These products are only to be used with the COBAS AmpliPrep instrument and the Cobas TaqMan Analyzer.
Detection of HIV-1 RNA is dependent on number of virus particles in specimen and may be affected by method of collection and patient factors (age,presence of symptoms, and stage of infections.
Mutation within the region of genome,(primer and probe) may result,even if rare.

16. Causes of Erroneous Results
Improper specimen collection, processing and transport.
Contamination from a Dnase and Rnase free room.
Improper mixing of reagents.
Reporting results when controls were not in normal range.
Not doing Quality Control on instrument.

17. Summary-1
The Cobas AmpliPrep/Cobas TaqMan HIV-1 Test is an in vitro nucleic acid amplification for HIV type 1 RNA found in human plasma.
The instrument is an automated specimen processing and the automated amplification and detection analyzer.
The machine is used to access patient prognosis by measuring the baseline HIV-1 RNA level or by measuring the changes in plasma HIV-1 RNA levels during course of treatment.

18. Summary-2
The test utilizes specimen preparation, reverse transcription and PCR amplification.
The process has a target amplification and a selective amplification.
The detection of PCR products shown by fluorescence are measured by intensity emission.